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Conversion of adenosine triphosphate (ATP) to the second messenger cyclic adenosine monophosphate (cAMP) is an essential reaction mechanism that takes place in eukaryotes, triggering a variety of signal transduction pathways. ATP conversion is catalyzed by the enzyme adenylyl cyclase (AC), which can be regulated by binding inhibitory, Gαi, and stimulatory, Gαs subunits. In the past twenty years, several crystal structures of AC in isolated form and complexed to Gαs subunits have been resolved. Nevertheless, the molecular basis of the inhibition mechanism of AC, induced by Gαi, is still far from being fully understood. Here, classical molecular dynamics simulations of the isolated holo AC protein type 5 and the holo binary complex AC5:Gαi have been analyzed to investigate the conformational impact of Gαi association on ATP-bound AC5. The results show that Gαi appears to inhibit the activity of AC5 by preventing the formation of a reactive ATP conformation., Competing Interests: The authors have declared that no competing interests exist.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gβγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells., Competing Interests: The authors have declared that no competing interests exist.

Adenylyl cyclase (AC) is an important messenger involved in G-protein-coupled-receptor signal transduction pathways, which is a well-known target for drug development. AC is regulated by activated stimulatory (Gαs) and inhibitory (Gαi) G proteins in the cytosol. Although experimental studies have shown that these Gα subunits can stimulate or inhibit AC's function in a non-competitive way, it is not well understood what the difference is in their mode of action as both Gα subunits appear structurally very similar in a non-lipidated state. However, a significant difference between Gαs and Gαi is that while Gαs does not require any lipidation in order to stimulate AC, N-terminal myristoylation is crucial for Gαi's inhibitory function as AC is not inhibited by non-myristoylated Gαi. At present, only the conformation of the complex including Gαs and AC has been resolved via X-ray crystallography. Therefore, understanding the interaction between Gαi and AC is important as it will provide more insight into the unknown mechanism of AC regulation. This study demonstrates via classical molecular dynamics simulations that the myristoylated Gαi1 structure is able to interact with apo adenylyl cyclase type 5 in a way that causes inhibition of the catalytic function of the enzyme, suggesting that Gα lipidation could play a crucial role in AC regulation and in regulating G protein function by affecting Gαi's active conformation.

Ocular albinism type 1 (OA), caused by mutations in the OA1 gene, encodes a G-protein coupled receptor, OA1, localized in melanosomal membranes of the retinal pigment epithelium (RPE). This disorder is characterized by both RPE macro-melanosomes and abnormal decussation of ganglion cell axons at the brain's optic chiasm. We demonstrated previously that Oa1 specifically activates Gαi3, which also signals in the Oa1 transduction pathway that regulates melanosomal biogenesis. In this study, we screened the human Gαi3 gene, GNAI3, in DNA samples from 26 patients who had all clinical characteristics of OA but in whom a specific mutation in the OA1 gene had not been found, and in 6 normal control individuals. Using the Agilent HaloPlex Target Enrichment System and next-generation sequencing (NGS) on the Illumina MiSeq platform, we identified 518 variants after rigorous filtering. Many of these variants were corroborated by Sanger sequencing. Overall, 98.8% coverage of the GNAI3 gene was obtained by the HaloPlex amplicons. Of all variants, 6 non-synonymous and 3 synonymous were in exons, 41 in a non-coding exon embedded in the 3' untranslated region (UTR), 6 in the 5' UTR, and 462 in introns. These variants included novel SNVs, insertions, deletions, and a frameshift mutation. All were found in at least one patient but none in control samples. Using computational methods, we modeled the GNAI3 protein and its non-synonymous exonic mutations and determined that several of these may be the cause of disease in the patients studied. Thus, we have identified GNAI3 as a second gene possibly responsible for X-linked ocular albinism., Competing Interests: The authors have declared that no competing interests exist.

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy.

Based on the recently described crystal structure of the β2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.

Background: Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction., Methodology: By using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis., Conclusion: Our results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.